ANALYTICAL CHEMISTRY
752 controlled by screw clamp P. I n place of P a float valve described by Carlsen and Borchardt ( 3 )may be used. By maintenance of a constant level in N the proper level is held in boiler A . Stopcock D may be eliminated if desired, but it is coiivenient for draining A' and the boiler at the end of a run. Vacuum line L is connected with manifold Q, and thus to lines F , all units being held at the same pressure. Air is alloLi-ed to enter into each boiler through E . These are To. 7300 capillary stopcocks, and a smooth-fitting piece of Nichrome wire can be inserted in the capillary above the stopcock to give the correct amount of air when the stopcock is wide open. I n place of this sliding-wire regulator, T joints may be sealed above the stopcock and a T cap made with a fine capillary constriction of the proper size to allow the right amount of air to pass. The small capillary must be protected from becoming clogged with fine particles of d w t from the atmosphere. The lines from R are also useful in draining the boilers and cleaning the still a t the end of a distillation. Ground joints and caps a t S are helpful during cleaning operations. Each boiler is served not only with its individual resistance heater, but also by a switch and a separate rheostat of 300-watt capacity and 5.48 amperes. With these controls the still can be run all day with very little attention other than an occasional adjustment of the boiler levels and rheostat. Slightly closer attention should be directed to flask A- to maintain the proper level there, as the even operation of all the boilers is partly dependent on this. The pressure a t which the still is run should be maintained at about 20 mm., mith the aid of any standard pressure regulator or manostat, and with the boilers a t 200" C. Cnder these conditions the air passing into the boilers (through R ) and over with the mercury vapor will oxidize any vapor of base metals. Hulett (6) has shown that under the proper conditions, with enough air passing over with the vapor, removal of base metals will be complete in one distillation. If the mercury is contaminated with gold or platinum, two distillations will be required for cleaning, while if the impurity includes silver, three successive distillations are required. I n this still the operator has the choice of bubbling air in one or all three boilers as the situation warrants.
The still may be cleaned by distillation of nitric acid in place of the mercury. With stopcock D closed, dilute nitric acid can be poured into each boiler through stopcocks R and the boiler heatere turned on as if for a regular mercury distillation. K i t h the condenser n ater turned off and L open to release any pressure, elevation of the temperature in the boiler nil1 send the acid vapor through the still and remove the mercury A final flush with dilute acid poured through R and S and several rinses with distilled water leave the still ready for drl ing and the next distillation. The stopcock shown on take-off line G is used without stopcock grease and is there only t o hold the pressure until G fills with mercury. \Then the still i s operating, the stopcock is left xide open. ACKhOW LEDG\IEVT
The author is indebted to G. Ross Robertson for his helpful Beniis suggestions in the preparation of this paper, to Russel \I-. for making the drawings, and to Cdwin 31. Griffith for hie help in designing and making the 0xidi7~r. LITERATURE CITED
(1) Bolton, Phys. Rev., 33, (1) 310 (1911). (2) Cannon, J . Chem. Education, 28, 272 (1951). (3) Carlsen and Borchardt, IND.ENG.CHEM.,-4x.i~.ED., 10, 94 (1938). (4) Friedrichs, Angew. Chem., 27, 24 (1914). (5) Hildebrand, J . Am. Chem. SOC.,31, 933 (1909). (6) Hulett, G. -4., Phus. Rev., 33, (1) 310 (1911). (7) Karsten, Instrumenlenk. 8, 135 (1888). (8) hleyer, Z . anal. Chem., 2, 241 (1863). (9) Moore, C. J., J . Am. Chem. Sac., 32, 971 (1910). (10) Patten, H. E., and Mains, S. H., J . I d . Eng. Chem.. 9, 600 (1917). (11) Tichers, E., Chem. Eng. S e i c s , 20, 1111 (1942). RECEIVED for review Xovember 21, 1950.
.4ccf?ixed August ? I , 1%l.
Chromatographic Separation of Metallic Chelates LELAND B. HILLIARD AND HENRY FKEISER University of Pittsburgh, Pittsburgh, Pa. LTHOUGH the chroniatographic technique has proved
A itself invaluable for many otherwise difficult analytical separations of complex organic compounds, its use in inorganic analysis has been almost neglected. Schwab and Jockers (6) have studied the adsorption sequence of metallic ions on alumina, and Hansen et al. ( 3 ) have employed chromatography in the separation of hafnium and zirconium chlorides from a methanol solution on a silica column. Benzoylacetone has been used as a complexing agent in the paper chroniatography of various metals ( 1 ) . 8-Quinolinol (8-hydroxyquinoline, osine) has been used as an adsorbent in columnar chromatography by Erlenmeyer and Dahn ( 2 ) and in paper chromatography by Laskomki and 11cCrone (4). The application of 8-quinolinol by these investigators might be considered as special cases, since chemical reaction (chelate formation) was involved in addition to chromatographic adsorption. Because an aqueouq phase was present, the process might be more akin t o partition chromatography. Although Belcher and Robinson in 1947 ( 5 ) suggested that, in view of the solubility of many metallic coniplexes in organic solvents, they could be subjected t o adsorption chromatography, to the authors' knowledge, no work has been published in which this technique has been utilized. I n the present \I ork, various metallic derivatives of 8-quinolinol were dissolved in an organic solvent (chloroform) and suhjected to the adsorption chromatographic process by being charged on a prepared silica column and being eluted with chloroform and chloroforni-ethyl alcohol mixtures. The chelates were found in niost cases t o form distinct, separably elutable bands. The application of this behavior to analytical separations and determi-
nations of metals makes possible the uqe of Chromatography as general analytical tool.
12
MATERILS
Activated Silica. l\lallinckrodt S o . 2847 silicic acid n-aA pre1 pared for use by heating it to 300" C. for 2 hours. Chloroform. Baker & Adamson chloroform was fractionally distilled before use and stored in brown bottles. Boiling point 59.5-60.5'. Ethyl Alcohol. Rossville Gold Shield absolute ethyl alcohol was used without further purification. 8-Quinolinol. Lemke Co. 8-quinolinol was twice recrystallized from alcohol-water mixtures. The recrystallized material was found to melt sharply at 72.5". \Then a chloroforni solution of this niat,erial was passed through a silica column, a brown residue was found. The chromatographically purified solution \yas stable for several days, as indicated by its chromatographic homogeneity. Solutions that' were allowed to stand for longer periods eshibited the brown decomposition zone. Hence, all &quinolinol used in this study was first subjected to chromato -1aphic purification within 3 d a r s of its use. $reparation of Chloroform Solutions of Metallic 8-Hydroxyquinolinates. The solutions of metallic 8-hydrosyquinolinatee in chloroform were prepared by extraction of the metal in aqueous solution by a chloroform solution of 8-quinolino1, rather than by direct solution of the solid 8-hydrosyquinolinate because, owing probably to the formation of hydrates and in some cases partially hydrolyzed products, these are not readily soluble in chloroform. A solution containing 10 mg. of the nietallic ion and 1 gram of sodium potassium tartrate was brought to a pH either 5.0 or 10.0 (as desired) by means of 0.1 N sodium hydrouide and extracted with IO-ml. portions of a 1% (by weight) solution of 8-quinolinol in chloroform until the color of the chloroform layer indicated complete extraction. No p H change ac-
753
V O L U M E 24, NO. 4, A P R I L 1 9 5 2 companied the extraction. The combined extract's W I Y dried by the addition of 2 grams of sodium sulfate. The sodiuni sulfate was washed with several portions of chloroform used to dilute to volume. I n all cases, the sodium sulfat,e held no color after washing with solvent. The following metals were easily extracted a t a pJI of 5.0: copper(II), nickel(II), cobalt(II), iron(III), aluniinum(III), galliuni(III), and indiuni(III), nhile a t p H 10.0 lead(I1) and Zinc(I1) and cadmium(I1) oxiiiates t ~ i ~ m u t h ( I I 1transferred. ) were of such limited solubility that even at, a pH of 10.0 a solution having a maximum concentration of approximately 0.5 mici~ogramper ml. could be obtained by extraction. T h e osimites of aluminum(III), galliuni(III), indium(III), ziiic (11), and cadmiuni(I1) exhibit fluorescence when exposed t o ultraviolet light,. This property served in determining the completeIIWP of extraction and also as a sensitive indication of the lack of retention of any of these chelates by the drying agent.
front of the zone to the effluent volume. RF values for S-quinolinol and the 8-quinolinates nere nieawred as a function of the amount of ethyl alcohol added t o the chloroform. Table I summarizes the data obtained. 8-Quinolinol could be eluted 151th pure chloioforni and v a y found t o have an RF y:iluc of 0.20. 7.0r
m
CHFOMAlDGFtAPHIC
ELUTION
OF M E T U I C DERIVATNES
CF 8- MDFOXYOUINOLNE
APPARATUS
Preparation of Silica Columns. An apparatus was assembled which made i t possible to make four chromatographic runs simultaneously. Dry, compressed air, regulated by a needle valve (of the kind used in the Tirrill burner), n-as conducted through a manifold into any or all of four 50-ml. burets which were charged with a loose slurry of from 8 t o 10 grams of activated silica in chloroform. The air pressure was maintained a t 50 cni. of mercury and the effluent rate was kept' a t 0.5 ml. per minute. C:HROMATOGRAPHIC EXPERIMENTS
Behavior of 8-Quinolinol and Metallic 8-Quinolinolates on Chromatographic Column. Because the chloroform solution of metallic 8-quinolinolates contained &quinolinol, two bands were obtained upon the chromatographic development of each of the 8-quinolinolates solutions. The 8-quinolinol was in all cases the first component eluted. While the original 8-quinolinol solution and the eluted 8-quinolinol solution were colorless, the 8quinolinol band on the silica column was of a bright orange color. This phenomenon is indicative of the structural change in the 8-quinolinol molecule, po~siblyof the kind
Copper 8-quinolinate gave a sharp, well defined dark-green zone; nickel 8-quinolinate formed a light green zone which mas rather diffuse; and aluminum 8-quinolinate, strangely enough, formed a zone of a striking greenish black hue. Like 8-quinolinol, the eluted aluminum 8-quinolinate solution was of the usual light yellow color. Iron(II1) and, on one occasion, cobalt(I1) 8-quinolinate gave h e t.0 two zones on the silica column. Both cobalt zones could be moved through t,he column when a 4% ethyl alcohol-chloroform solution was used as a n eluant, the faster, colored reddish brown, moving about' twice as fast as the other which was colored yellow brown. The iron zone rolored greenish black zone could be moved easily through the column, but, it was impossible to move the reddish black zone through. Both zones tvere shown t o be free of iron(I1) so that reduction was not causing the appearance of the two iron zones. The second, reddish black zone was also proved not t o be iron tartrate, chloride, or hydroxide. Possibly these zones indicate the presence of isomeric forms of iron oxinates. Adsorption Sequence of Metallic 8-Quinolinates. I n these experiments, in which 50-ml. burets were employed as columns, 1 cm. on the column is equivalent t o 1 ml., so that RF can be conveniently measured a8 the ratio of the dist,ance moved by the
EFFLUENT VOLUME 1 % ALCOHOL IN CHLOROFORM, ML.
Figure 1 The Rp values listed here are prol~ahlysignificant to better than 0.05. As can he seen from the table, the addition of ethyl alcohol t o the eluant increased the rate of movement of the 8-quinolinates zones up t o the point where a t about 6% ethyl alcohol the 8quinolinate was completely displaced by t,he eluant. The atlsorption sequence of metals bears no relation t o the order of stabilities of these chelates. While bismuth and lend oxinates could be easily extracted into chloroform solution, they formetl extremely diffuse bands on the silica column and thus RF values for these two could not be measured as were those listed in Table I. By passing lead quinolinat,e through the column in the p e s ence of nickel quinolinate, the lead band was found t o follow that of t.he nickel. By studying a mixture of bismuth and nickel and aluminum quinolinates it, was found that the bisniuth oxiii:tte and nickel. Hence, ior hand falls between those of alu~ni~iuni quinolinates in chloroform, the adsorption sequence i.; 8-quinolinol, copper, cobalt, iron, aluminum, bismuth, nickel, and lead quinolinates in order of increasingly strong adsorptibn hy silica.
Table I.
RF Yalues for IIetallic Quinolinates on Silica
CzHaOH in hluant, '7, 0 I >
b
Cii(I1)
Qiiinolinate Co(II)a Fe(III)b AI(II1'
___
SIUI)
0 06
Reddish brown band. Greenish black band.
.4 mixture of copper, nickel, and col)alt quinolinates n-as foulid to be separable into three distinct fractions as predicted by the RF values, when 1% ethyl alcohol in chloroform was used as eluant (Figure 1). I n this run, 0.12 mg. of each metal was eluted following the procedure of extracting, drying, diluting to volume, :ml charging a n aliquot to the column. The orange 8-quinolinol zone was removed completely by chloroform elution (this band not shown in Figure 1). The eluant was then changed to 1% ethanolic chloroform. Effluent samples were analyzed spectrophot ometrically. The metals were recovered t o within 0.02 mg.
ANALYTICAL CHEMISTRY
754
LITERATURE CITED
While not fully investigated, it is believed that a difference in
RF values of 0.05 to 0.1 between two components would he suffi-
(1)
cient to permit a complete separation. Kith substances whobe bands were especially diffuse the difference in RF values nould naturally have t o be larger.
(2)
(3)
(4) ACKNOWLEDGMEhT
T h e authors gratefully acknowledge the financial support of the Atomic Energy Commission. This noik was performed under Contract 8T-(30-1)-860.
(5)
(6)
Elbeih, McOmie, and Pollard, Discicssion~.Faraday SOC.,KO.7, 183 (1949). Erlenmeyer and Dahn, Helr. Chi7rz. Acta, 22, 1369 (1939) Hansen, Gunnar, Jacobs, and Simons. J . Ant. Chem. SOC., 72, 5043 (1950). Laskowski and McCrone, Abstracts of 119th Meeting, . h i , CHEM. Soc., Cleveland, Ohio, p. 19B, 1951. Robinson, Melallurgia, 37,45 (1947). Schwab and Jockers, Angezc. Chmu., 50, 546 (1937).
RECEIVED for review August 22, 1951. Accepted October 31, 1931. Contribution 839, Department of ChemiPtrj-, Vniversit)- of Pittsburgh.
Alpha-Keto Acids in Blood and Urine Studied by Paper Chromatography D i V I D SELIGS0,N'
AND
BERN.4RD SHAPIRO
George S. Cox Medical Research Institute, University of Pennsylvania, Philadelphia, Pa.
l H E accurate and specific determination of keto acids in blood and urine has been inipossihle because of the lack of bpecific, sensitive, stoichiometric reactions. Cavalliqi and coworkers (3, 4) converted the keto acids of blood and urine to their 2,4-dinitrophenylhydrazones,sepamted this group on papc'r chromatograms, and measured 'the isolated hydrazones colorimetrically. The method reported here is B modification of Cavalh i ' s , which in the author's laboratory was easier to execute. Previously described methods ( 2 , 5 , 7-13, 18) for pyruvic acid, the main a-keto acid of blood and urine, have lacked the specificity of the method mentioned above, although an approach to specificity was made by Friedemann and Haugen ( 8 ) , \+-hotook advantage of the difference in solubilities of the dinitrophenylhydrazones. Other workers, using diatomaceous earth colunins ( 1 4 ) and alumina columns ( 6 ) , have recently reported the separation and measurement of keto acid dinitrophen>.lhydrazones. The latter method (6) is not sensitive enough for the determination of very small quantities. I n the early experiments of this laboratory, separation of keto acid hydrazones was attempted by fractionating on a silica gel column and chromatographing the fractions on paper. .4lthough this method gave a resolution of keto acids of urine, coincidental studies Ivith paper chromatography alone, which gave 'complete and specific. resolution, indicated the use of this simpler method. The usefulness of the present niethotl lies in its specificity for individual keto acids. I t has enabled the identification and quantitative estimation of two intelmedintes of carbohydrate metabolism in blood and urine nntl the demonstration of a t least two other unidentified keto acids normally present in urine. The method is reliable for samples roiitaining R S little as 5 mic.rograms or less of pyruvic or a-ketoglutaric acids. hI ETHODS
Reagents. Sulfuric acid, 0.66 S. Aqueous sodium tungstate solution, loc ;. Sodium carbonate 1 N . Sodium bicarbonate, 1 AT. Sodium hydroxide, 1 N . IIydrochloric acid, 6 1%;. Chloroform, containing 20", ethyl alcohol. Acetone. 2,4-Dinitrophenylhydrazine Solution. This contained 5 nig. of 2,l-dinitrophenylhydrazine per nil. of 6 S hydrochloric acid. The reagent was dissolved with gentle heating on a steam bath and constant su-irling. Solvent for Chromatography. One part of butanol and 2 parts of 1 S sodium bicarbonate were shaken in a separatory funnel for 2-minute periods several times during the course of the esperinient, to equilibrate the solvent with the water. 1
Present address, Army Medical Center, XTushington 12, D. C.
Standards. Pure sodium pyruvate and or-ketoglutaric acid were dissolved in water t o give a single solution containing 0.25 micromole per ml. of each. When 1 ml. of this standard was diluted t o 50 nil. with water, this was equivalent to 5 ml. of blood or urine containing 0.05 micromole of pyruvic acid and 0.05 micromole of a-ketoglutaric acid per nil. (or 0.44 and 0.73 mg. per 100 ml., respectively). Filter Paper for Chromatography. Strips of Whatman KO. 1 filter paper, 10 by 51 cm., were used. These were previously wetted with 1 N sodium bicarbonate (pH 8.2) and dried. Variations in pH as well as in the concentration, cations, and anions of the buffer were tested. It was found that 1 iV sodium bicarbonate produced the best resolution of the hydrazones. The papers were hung in a trough which fitted across the top of a cylindrical jar. The jar had a rubber collar, which permitted it to be tightly covered with an appropriate enamel pan. Procedure. Ten milliliters of freshly drawn venous blood were immediately added t o 70 ml. of water and 10.0 ml. of 0.66 S sulfuric acid. This was shaken briefly, 10.0 nil. of lOyo sodium tungstate were added, and after further shaking the misture was filtered. Removal of the protein in this manner does not interfere with keto acid recoveries. I n recovery experiments the standard was added directly t o xhole blood. Standards, prepared as above, were treated like the blood filtrates. Urine, diluted 5.00 nil. t o 50 ml., was run similarly. T o 50.0 ml. of sample (blood filtrate, diluted urine, standard, or blank j 2 . 0 0 ml. of 2,4-dinitrophenylhydrazine reagent wert added. The mixture was a l l o w d t o stand for 30 minutes at 25 Cy. It was then transferred to a 125-ml. separatory funnel and the hydrazones were extracted from the aqueous solution with three 15-ml. portions of chloroform-ethyl alcohol solvent. In the case of urine i t was necessary to centrifuge for 5 minutes after extraction in order t o separate the emulsion that formed. The combined chloroform layers were extracted with 15 nil. of 1 .V sodium carbonate and the chloroform was discarded. After the sodium carbonate containing the hydrazones had been washed with 10 ml. of chloroform-ethyl alcohol, the carbonate solution cidified with 5 ml. of 6 S hydrochloric acid. The hydrazones were then extracted from the aqueous layer with three successive portions of chloroform-ethyl alcohol, 10, 5, and 5 ml., respectively. The three extracts containing the keto acid derivatives were combined and evaporated under a gentle air blast. Evaporation required 30 t o 45 minutes. The total residue from the chloroform-carbonate-chloroform estmction, consisting chiefly of the keto acid hydrazones, was quantitatively transferred t o paper by dissolving it in 0.3 ml. of acetone and applying the solution to the top of the paper strip ti,ansversely as an 8-em. streak. The evaporation vessel was rinsed ivith three 0.2-ml. additional portions of acetone, each of which was applied t o the paper along the original streak. .4 gentle blast of warm air accelerated the drying. Aftel, this transfer the paper was suspended in the glass cylinder, PO that the top edge dipped into the trough while the bottom edge of the strip hung 2 cm. above the bottom of the cylinder. The bicarbonate solution, after equilibration with butanol, was placed in the bottom of the cylinder, which was tightly covered with an enamel pan. After an equilibrium period of a half hour or more, the butanol was added t o the trough. The butanol was allowed t o move down the paper overnight (14 hours), achieving complete resolution of the keto acid hydrazones. The paper was then removed from the apparatus and air-
